Purpose :
Most pigmented lesions of the conjunctiva are benign, such as nevi and primary acquired melanosis (PAM) without atypia. However, certain pigmented lesions are more prone to malignant progression, such as PAM with atypia. Currently, single immunohistochemical (IHC) staining with Melan-A (MART1, melanocyte antigen) and microphthalmia-associated transcription factor (MITF) identify all melanocytic cells, while human melanoma black 45 (HMB45) and Ki67 identify potentially proliferative cells. However, these single stains may not always identify cells that are both melanocytic and proliferative. Our study implements a novel double-staining technique to identify the most sensitive and specific combinations of Melan-A, MITF, Ki67, and HMB45 aiming to better diagnose atypical melanocytic lesions that are more prone to malignant progression.

Methods :
Eight specimens of conjunctival melanoma and PAM with moderate to severe atypia were obtained from the ocular pathology registry of the University of Ottawa. Sections from each formalin-fixed paraffin-embedded specimen were double-immunostained with a combination of a cytoplasmic marker (anti-Melan-A or anti-HMB-45), and a nuclear marker (anti-MITF or anti-Ki67). These specimens also were processed using 3,3-diaminobenzidine substrate for nuclear stains and the vector red method for cytoplasmic stains. Each slide was analyzed by light microscopy and provided 0-100% staining positivity, as well as a 0 to 4+ score, for each nuclear and cytoplasmic component. Eight specimens of normal conjunctiva and conjunctival nevi served as controls.

Results :
Melan-A and MITF are strong markers for all melanocytic cells, whereas Ki67 and HMB45 provide a variable response for identifying proliferative cells. However, when combined with HMB45, a double-stain utilizing Ki67 is most effective at differentiating between malignant and benign lesions. The combination of HMB45 and MITF is best at identifying all melanocytes, while demonstrating the subpopulation of atypical cells (p<0.05). The 2 other combinations also are of diagnostic assistance.

Conclusions :
Our study is the first in the ophthalmic literature to utilize an IHC double-staining approach to differentiate between malignant and benign melanocytic lesions of the conjunctiva. Our results should aid in the more complete histopathological diagnosis of conjunctival melanocytic lesions.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.